Genome sequences contain the information needed for the development and function of a given species. Exploring conservation of DNA sequence between species is a tool for identifying critical code that is likely to be important for functions shared between species. Genomic information is deployed by transcription and differential expression may well be important for phenotypic distinctions among closely related species. Comparative transcription has not been extensively explored. We completed and published deep sequencing experiments (RNA-Seq) to probe expression in several of the newly sequenced Drosophila species. These expression data provided strong functional validation of the Drosophila melanogaster annotation, and demonstrated methods to validate gene models in other species, including humans. The information content of the genome is increased by alternative processing of primary transcripts. Genome-wide analysis of alternative splicing is prone to errors. We have undertaken computational efforts to improve the methodology in this arena. We have begun to explore how various genes regulate splicing. How gene interact in networks is an important underpinning for understanding development, physiology, and disease. We are looking at the transcriptional response to altered gene dose in aneuploid tissue culture cells, during X chromosome dosage compensation, and in a series of flies heterozygous for a tiling path of deletions (DrosDel). The DrosDel work is being expanded in terms of breadth of genome coverage and by the addition of tissues. We are mapping the coherent propagation of dosage effects elsewhere in the genome, and using the data to machine-learn network models to explain the observed patterns of gene expression. We continue to pilot the use of small compounds and FDA approved drugs, RNAi reagents, Crispr, and genetic background, as tools to perturb gene expression networks. These data are providing important validation of connectivity maps and will allow us to add directionality to network edges, as well as help us understand how different individuals respond to common external challenges. DMRT transcription factors direct gonadal development in essentially all animals, but little is known about the gene networks they regulate. We are working on the function of the Drosophila DMRT (DSX) and are particularly interested in the identification of functional DSX transcription factor target genes. We have previously used ChIP-Seq and DamID-Seq to determine the in vivo occupancy of DSX using tagged and native proteins in tissue culture cells and in Drosophila tissues and examined expression profiles in wild type and mutant conditions (especially those where we change the isoform of Dsx being expressed and measure the response over time) by RNA-Seq. We are also performing knockdown experiments to test for the function of candidate DSX targets using RNAi (UAS-shRNAs driven by Dsx-Gal4 and other Gal4 lines). Finally, we are asking if these target genes show dominant genetic interactions with mutant Dsx alleles. We have selected potential DSX targets skull, small ovaries, and broad for detailed phenotypic follow-up to determine the function of these genes in the cells that are in direct contact with the germ line cells in the female and male gonads.